CA1101318A

CA1101318A - Diagnostic agent with a new stabiliser

Info

Publication number: CA1101318A
Application number: CA300,069A
Authority: CA
Inventors: Walter Rittersdorf; Hugo Tiedemann; Wolfgang Werner; Hans Wielinger
Original assignee: Boehringer Mannheim GmbH
Current assignee: Roche Diagnostics GmbH
Priority date: 1977-04-09
Filing date: 1978-03-30
Publication date: 1981-05-19
Also published as: SE442680B; SE7803889L; SU1457819A3; NL7803722A; FR2386823B1; ATA248578A; GB1571363A; JPS5643238B2; FI781015A; HU177174B; DE2716060A1; BE865701A; AT361132B; FR2386823A1; IT1095396B; AR215172A1; JPS53126996A; DE2716060C3; US4220713A; DD141361A5

Abstract

ABSTRACT OF THE DISCLOSURE

1-Arylsemicarbazides are provided as stabilizers for oxygen indicators functioning as chromogen in diagnostic agents employed in the detection of glucose in body fluids, such as urine and blood; and the detection of blood in body materials such as urine, faeces and liquors; the stabilizers enable the manufacture of reliable test strips for rapid testing in medical diagnosis; the 1-arylsemicarbazides have the formula:
Ar-NH-NH-CO-NH2 wherein Ar is an aryl radical which may be substituted by alkyl, alkoxy or halogen.

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Description

a3~8 The present invention is concerned with an improved diagnostic agent for the detection of hydroperoxides or of substances which react with the liberation of hydroperoxides, as well as of peroxidase or of peroxidate-active substances, in bcdy fluids, which agent contains an oxidation indicator as a chromogen. More especially the invention is concerned with l-arylsemucarbazide stabilizers for the oxygen indicator.
The diagnostic agent according to the present invention is especially suitable for the detection of glucose in urine and blood, as well as for the detection of blood in urine, faeces and liquors.
Rapid diagnostic agents have recently achieved very considerable impo~tance in medical practice and in clinical laboratories and especialLy rapld tests based upon absorbent carriers. The absorbent carriers, which are usually paper, are impregnated with the reagents necessary for the detection reaction and show, after simple immersion into b~dy fluids, a colour reaction when the substance to be detected is present.
Rapid tests which have achieved great importance in medical diagnosis are, in particular, rapid tests for the detection of glucose in urine and blood, as well as for the detection of blood in urine, faeces and liquors.
These tests are based upon the following principle:
in the case of the rapid tests for the detection of glucose in body fluids, for example, the glucose is oxidised by atmospheric air in the presence of a catalytic amount of glucose oxidase (GOD) to give gluconic acid, t~e atmospheric oxygen thereby being reduced to hydrogen peroxide. The hydrogen peroxide ~ -~
thus formed oxidises an oxidation indicator, this oxidation being catalyzed by peroxidase or a peroxidate-active substance to give a coloured material, the depth of colour o~ which is a measure of the amount of glucose present.

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In the case of the detection of blood, the peroxidate properties of haemoglobin are utilised in order to oxidise the oxidation indicator with the help of a hydroperoxide.
Oxidation indicators or the above~mentioned rapid tests are generally known and fall within certain classes of compounds. Particularly usual are representatives of the benzidine series and heterocyclic azines, as well as phenolic bodies and especially the components of guaiac resin.
Due to their very nature, oxidation indicators are easily oxidisable substances, Therefore, it is not surprising that they can be oxidised by air, especially in the light.
For these reasons, potentially useful oxidation indicators cannot necessarily be used for rapid diagnostic agents because their storage stability is very limited.
Therefore, the problem exists of finding appropriate protection for these oxidation indicators in order that they can be used for rapid diagnostic reagents without he sensitivity of the test being disadvantageously influenced by the amount of stabiliser added. The substances which ensure such a protection must, in turn, be sufficiently stable and must not uncontrollably or drastically change the sensitivity of ~ ;~
the test papers.
Surprisingly, we have now found a group of 1-aryl-semicarbazide compounds which bring about the desired effect in an out~tanding manner.
Thus, according to the present invention~ there is provided a diagnostic agent for the detection of hydroperoxides or of substances which react with the liberation of hydro- ~ -peroxides or of peroxidase or of peroxidate-active substances in body fluids, u~ing a stabilised oxidation indicator and using the reaction of a hydroperoxide or of a peroxidate-active : ~ .

- 2 - - ~
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substance with an o~îdation indicator and evaluating the coloration produced by the reaction, wherein the stabiliser used is a l-arylse~icarbazide of the formula (I):
Ar-NH-NH-C0-N~I2 (I) wherein Ar is an aryl radical unsubstituted or substituted by alkyl, alkoxy or halogen.
If desired, the diagnostic agent according to the s present invention can also contain at least one member selected from buffers, wetting agents, thickening agents, 10protective colloids and complex formers.
By an aryl radical, there is preferably to be under-stood a phenyl or naphthyl radical; the alkyi and alkoxy radicals suitably contain 1 to 6, preferably 1 to 4 carbon atoms, the methyl, ethyl, methoxy and ethoxy radicals being especially preferred. Halogen can be fluorine, chlorine or bromine, chlorine being preferred.
- According to another aspect o~ the invention there is provided a diagnostic test strip for rapid testing of body materials which comprises an absorbent substrate impregnated 20with an oxygen indicator and a stabilizer of formula(I), as defined above. -Accordiny to a further aspect of the invention there is provided a method for the detection~ of occult blood in faeces which comprises appl~ing a sample of the faeces to a first side of a test strip of the invention, applying hydrogen peroxide dropwise to a second side of the test opposite the first side, and evaluating any colouration as an indication of the presence or absence of peroxidate-active blood components in the sample, In another aspect of the invention there is provided a method for the detection of blood in body fluids in which a .

IL3~ :

test s~xip of the invention impregnated with a hydroperoxide is employed, In this case the dropwise addition of hydrogen peroxide is omitted.
In still another aspect of the invention there is provided a method or the det~ction of glucose in body fluids in which a test strip of the invention impregnated with glucose oxidase and peroxidase or a peroxidate-active substance is e~ployed.

It will be understood that the components of the diagnostic agent are to be employed in amounts effective to meet their function. Thus the oxygen indicator should be present in an amount effective to produce a perceptible coloration on o~idation; and the l-arylsemicarbazide stabilizer should be employed in an amount effective to stabilize the ~
oxygen indicator against premature oxidation by atmospheric ~ ;
oxygen, especially in the light~ Suitable amounts can be readily detaxmined by persons skilled in the art and especially suitable and convenient amounts are described herein.
It is surprising that the l~arylsemicarbazides tI) possess such outstandingly favourable properties. It was admittedly known that these compounds, as derivatives of hydrazine, have reducing properties; however, it has been shown that reducing agents are not automatically suitable for the purpose of stabilising oxidation indicators without sub-stantially influencing the ~ensitivity thereof. Thus, for example, the parent material of the series, i.e~ semicarbazide, i9 not suitable for this purpose. -~;~
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For quite a long tirne, other reducing agents, for example, ascorbic acid, hydroquinone and other compounds, have ~een described as "modifiers" of the sensitivity of reactions (cf~ German Patent 1,129,003 M. H. Cook, isswed May 3, 1962 and 2,555,704 ~, Hochstrasser, issued June 24, 1976, and U.S. patent 3,008,879 E. K. Harvill, granted Movember 1~, 1961), but none of these compounds has been used in practice because, on the one hand, they are too unstable and, on the other hand, as a result of this instability during the time up to use, the sensitivity of the test is disadvantageously changed. Known anti-oxidants, ~or example, nordihydroguaiaretic acid, N-phenyl-naphthylamine and mercapto compounds, as well as the compounds mentioned in the aforementioned U.S, patent 3,008,879 and German Patent No. 2,S55,704, are not very suitable since they are either ineffective for the indicators used in rapid diagnostic agents or they are too unstable or they have too ~; strong an influence on the reactivity. ~`
The advantageous use of the stabilisers accordin~ to the present invention in test strips which contain oxidation indicators as chromogens is further explained in the following:
For the dstection of blood or glucose, o~tolidine has hitherto been used in rapid tests as indicator. However, this carcinogenic substance has recently been replaced by -

3,3',5,5'-tetxamethyIbenzidine tTMB) (cf~ German Patent No.
2,460,903 W. G~thlein, issued June 24, 1976)~
Unfortunately, test papers with this indicator are, as in the case of all benzidine bodies, light-sensitive~ Even after leaving in the light for a few minutes, they become dis- ;
coloured. An additional disadvantage is to be specially o~served in the case of test papers for the detection of glucose, namely, the ease with which the indicator is oxidised in the air and "' .
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especially in the presence of atmospheric moisture. This pheno~non is very disadvantageous because it is precisely indicators for the detection of glucose in urine which come into the hands of lay persons for precautionary investigations.
Test papers which contain the l-arylsemicarbazides (I) to be used according to the present invention surprisingly do not exhibit these disadvantages. Furthermore, the amount of l-arylsemicarbazides (I~ used in the test agents can be employed within wide limits of tolerance without substantially and disadvantageously influencing the sensitivity of the test agent.
In addition, the l-arylsemicarbazides used according to the present inventîon are generally use~ul for glucose-tests and for tests for the detection of blood in faeces which contain, as indicators, heterocyclic azines, such as are described in German Patents 1,648,840 H.-G. Rey, granted JUne 4, 1970 and 1,917,996 H.-G Rey, granted September 6, 1977.
1-[3-Alkylbenzthiazolone-(2)]-2-~1-phenyl-3-methyl-

4~ethyl-1,2,4-triazolone-~5)]-azines and 2,2'-azino-di-Cl-ethyl-quinolinone-~2)-disulphonic acid-(6)], have proved to be especially advantageous, They here also provide a greater stability towards discolorations brought about by light and air which, in turn, can easily give falsely positive reactions.
However, test papers for the detection of blood in faeces preferably contain guaiac resin or a purified fraction thereof, namely guaiaconic acid A, with the specific i~
extinction of El%Cm at 600 nm of at least 200, deterl~ned by ~-the reaction with peroxidase and hydrogen peroxide, according to co-pending Canadian Patent Application of W. Guthlein et al (corresponding to West German Patent Application P 27 16 061v8, filed April 9, 1977), filed ~imultaneously herewith. Such test ,' 3~

papers also have a tendency to become blue coloured in air and light, which can result in dist~rbances due to falsely positive reactions, Mere, too, the l-arylsemicarbazides (I) used according to the present invention prevent this disturbing and undesired blue coloration.
The test papers according to the present invention can be produced by known methods in which, to a particular formulation, there is added a l-arylsemicarbazide (I) to be used according to the present invention (cf. the aforementioned German Patents 1,648,840; 1,917,996 and 2,460,903).
In general, the hydroper~cides used in the test papers or strips can be of any of the conventional respresentatives of this class of compounds, which are relatively non-volatile, for example, tert.-butyl hydroperoxide. In particular, there have proved to be useful the so:lid compounds 2,5-dimethylhexane-2,5-dihydroperoxide, tetraline hydroperoxide and diisopropyIbenzene dihydroperoxide, as well as liquid representatives, for example, diisopropyl-benzene hydroperoxide, cumol hydroperox1de, ~-menthane hydroperoxide and pinane `~
hydroperoxide.
It will be under~tood that the hydroperoxide in the test strips employed for the detection of blood in body fluids, is impregnated in the test strip in an amount effective to oxidize the oxygen indicator to produce a perceptible coloration, in the presence of peroxidate-active substances found in blood.
In this respect the hydroperoxide can suitably be employed in an amount of from 0.5 to 5 g~ and preferably 1 to 3 g. per 100 ml. of impregnation solution.
In the~embodiment in which the presence of occult blood in faece~ is to be detected, the hydroperoxide is most : : :
suitably hydrogen peroxide and is not incorporated in the test strip but i~ applied separately, after application of the 3~l8 faeces sample to the strip, suitably in solution, for example, an alcoholic solution.
In test strip~ of the invention for the detection of glucose in body fluids there is additionally employed glucose oxidase and peroxidase or a peroxidate-active sub-stance. The glucose o~idase catalyzes the oxidatio~ of glucose by atmospheric oxygen, and should, therefore, be present in -a catalytic amount to achieve this function, The peroxidase or peroxidate-active substance catalyzes the ~xîdation of the oxygen indicator by hydrogen peroxide generated in the aforementioned oxidation of the glucose, and should, therefore, he present in a catalytic amount to achieve this function.
As buffers, there can be used, for example, citxate, ~ `
phosphate, phthalate ox succinate buffers.
It is also advantageou~ to add to the formulation `-: . :
small amounts (about 0.05 to O.S g. per lOQ ml.) of a complex former, for example, sodium metaphosphate or an àlkali metal salt o~ ethylenediaminé-tetraacetic acid, in order to avoid falsely positive reactions due to the presence of traces of metals. The complex formers can also act as buffers.
Since, dué to the presence of relatively large amounts of water-~oluble substances, the test papers can have a tendency to bleed, it is advisable to add thickening agents '~
to the formulation, for example, methyl cellulose or especially gelatine, in amounts of about 0.5 to 5 g, per 100 ml. In some ~
cases polyvinylpyrrolidone has also proved to be useful~ -A5 wetting agent, it is preferable to use a long-chained organic sulphate or sulphonate, for example, sodium dodecyl-benzene-sulphonate, dioctyl-sodium sulphosuccinate ;
or sodium lauryl sulphate, For the production of the test papers according to th~ present invention, absorbent test papers, for example, filter paper, cellulose or synthetic resin fleeces, are impregnated with solutions of the reagents in readily volatile solvents~ This impregnation is preferably carried out in two stages, the co~positions of the individual solutions depending upon the particular rapid test to be produced, For the production of a rapid test for the detection of blood in faeces with the use of guaiac resin or guaiaconic acid A, it is advantageous when the hydro-peroxide, in this case hydrogen peroxide, is not impregnated into the carrier but rather is added dropwise after application of the faeces.

3~8 The l-aryl~Qmicarbazides used according to the present in~ention can be added in amount~ of fro~ll about ~-

5 to 900 mg. per 100 ml. of impregnation solution. .:
&enerally, the amount used depend~ upon the pu~pose for ; ~:
w~ich the addition i~ made. ~-The l~arylsemicarbazide~ used according to the present i~vention are either Xnown or can be prepared ::
analogously to known proc sses (see 0. Widman, Ber~, 26, 2613/1~93, J.TA Hewitt, J. Chem. Soc., ~09/1891)~
q~he following ~3xamp1e~ are given for the purpo~
of illu~trating the present invention, the percentages therein being ~y weight and the abbreviation K-EDTA u ed therein meaning the potassium salt of ethylenediamine- :
tetxaacetic acid.
Example 1. . . , _e~ ~oc~
.
The synthesis start~ ~rom appropriate aromatic ~ .
amines which are diazotised to give the corre~ponding diazonium compounds. ~he diazonium salt is converted into the corresponding hydrazine in hydrochloric acid solution by means of sta~nous chloride. Reaction of the:
hydrazine hydrochlorid or of ~he free hydrazine base in glacial ~cetic acid with potassium cyanate give~ the desired 1-aryl3emicarbazide in good yield.
In the following ~able3, there are set out some of the l-arylsemicarbazide~ which ca~ be used according :
to the present invention, ~ome of which are new. Those which are ~ew are indicated with an a~terisX.

3~ 51 T A B L E

Ar-NH-~-C-NH~

Ar_ _¦ m-p- ¦ ~iterature reference Cl 1~8 ~ 170 ~ ;~

151 ~ 153~ J~To Hewitt, J. ChemO SO
C1 ~ 868/1873 :

228 231 J~T. Hewitt, J. Chem. Soc., . 209/1891 ~ 159~ J.V. Janowski, J~ Reimann, ~ C 3 18Z 8er. 21 2Z1fl88 ~CH3 135 . '~

231 A~ Pi~ner, Ber. 21, 1222/18~8 170~173 Commercial product of _ _ ._. E. Merck ` ~

3~8 :
Test for the detection of occult }~lood in faece~
Filter paper (Schleicher ~ Schull 597 ~Ind. ) i~3 succe:3sively impregnated with the following solutions and dri ed:
Solutîon lo 0.34 molar K-EDTA buffer, pH S.5 10 ml~
polyvinylpyrrolîdone 300 my.
di~ti~led water ad 100 ml.
Solution 2:
guaiaconic a~id A, ~lYcm ~ 260 at 600 nm 130 mg.
l~phenyl~emicarbazide .. 65 mg.
ac~tone ad 100 ml. -In-~tead of l-phenyl~emicarbazide, there can ~e used ;
an ~quimolar amount of any of the o~her ~ompounds ~
mentioned in Exa~le 1. ` ~ -qhe concentrations of the l-axylsemicarbazides can vary within the range of ~ 30~ by weight, referred to 2 0 the above-given amount~, without the sensitivity o the detection reaction being markedly influenced or without ~he stabili~ing effe~t being changed................... ~:
~e~t paper~3 ~ich have been produced by the process de~cribed in thi~s Example are stable towards the influence~ of light and air and are out~3tandingly useful :Eor the detection of pathological amount~ of blood in faeces. For this pu~pose, faece~ are appïied to t}le test paper, allowed to dry and developad fro~ ~the rear ide with an alcoholic solution of hydrogen peroxide. If pathological æmounts of ~1QOd are present, then a ~right ' ~, ; *trademark blue coloured 20ne result~ which radiate~ out from the ~ample.
Exam~le 3.

*
Filter paper 'Schleicher ~ Schull 597 ~F-Ind.
~uccessively impr~gnated with the following solution~
and then dried:
Solllti~n 1:
0.34 molar K~EDTA buffer, p~ 5.5 10 ml~
polyvinylpyr~olidone 300 mg.
distilled wat~r ad 100 ml.
9O1ution 2:
guaiaconic acid A, Ell%Cm = 260 ~: :
at 600 nm . 130 mg. ~ :
antiox1dant 60 mg. ~ :
ethanol ad lOO ml. ~ -A~ antioxidant~, there are u~ed 2,6-di-tert.-~utyl- .
.
~-cresol, gallic acid, gallic acid ester9, pyrocatecholO
2,6-di-tert.-butyl-phenol, 2,6-dihydroxybenzoic acid, ~ `
gentisic acid, nordihydrcguaiaretic acid, ~-phenyL-a~
naphthylamine, triphenyl phosphi~e, benzimidazole and 2,2-bis-(~-hydroxyphenyl) propane.
The papers ~o produce~ are compared wi~h papers which have been producad according to Exampla 2 and ~ tested for their u~efulness or the detection of patho~
logical amount~ of blood in faeces. All antioxidant~ act `:
in ~uch a manner that they suppress the det~ction of pathological amounts of blood in faeces.
If th~ concentration of the antioxidant in ~he formulation is reduced to such a~ extent that there i~ ~
*trademark . . :

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again obtained a sufficient 5en9iti~ity of the test, then these subqtance~ lose their stabilising action Exam~le 4O ~
Detection of blood in faeces. ~-Filter paper (Whatman ~o.l~ i3 successively impreg- -~
nated with the followQng ~olutions nd then dried~
Solution 1:
O.34 molar K-EDTA buffer, pH 5~010 ml.
dioctyl sodium 4ulpho~uccinate 500 mg.
methanol Z0 ml.
distilled water 80 ml.
Solution 2:
__ .
3,3'~5,5'-tetramethylbeDzidine 40Q mg.
l-phenylsemicarbazide 100 mg~
acetone 100 ml.
A test paper produced in this manner fully ~atisfie~
the r~quirement3~whlch are d~manded of a test for the detection of ~athological amounts of blood in faecas.
~ .
~ter th~ development o a sample of faeces applied to the te~t paper by~mean~ of an alcoholic ~olution of hydrogen poroxide, pathological amounts of blood are dete~ted. A green colour forming round the ~mple indicates the blood. The test paper is sufficiently stable to~ards light and at~ospheric oxidation~
A test paper which has been produced according to the ab~ve formulation but without the u~e of the l-aryl-semicarbazide acaording to tho present invention, on the : ~:
other handO becomes.blue-green coloured within a short. ~:~
period of time in iight and air~ It ~an no longer ~e u~ed for diagnosti~ purpo~eq.

*trademark - 14 _ 3~3 Example 5.
Detection of_blood in faeces Fil ter paper ~ Schleicher & Schull 59~ ~-Ind~
successively impregnated w~th the follo~fing solutions and then dried:
Solution 1:
Corresponds to Solution 1 of Example 4 Solution 2:

~ 3-ethylbenæthla2Olone-(2)~-2-[1-phenyl~3~methyl-4-ethyl~1,2,4-triazolone-(5)~-a~.ine 100 mg. -l-phenyl~emicarbazide 70 mg.
acetone 100 ml~
Such a paper ha~ the ~ame propertie~ a~ one producea according to Example 3, the resulting reaction colour i5 blue. Papers w~ich have been produced without ~he addition of the l-arylsemicarba~i:de according to the ; :
present invention become discoloured even during the ~:
detection reaction in such ~ manner that,:even in.the case 3f ~aeces o~ healthy subject~, a falqely positive reaction can be ~imulated.
~ .
, .
Te~t pa~er for the de _ tion o blood_in_urine or liq~r.
Filter paper (Schleicher & Schull 23 SL~ is suc~essivsly impregnated with the followîng ~olutions ;~
and then dried:
Solution 1 _ . . . .. . .
~ . ~
-1~, 2 molar sodium citrate buffer, pH 5. ~5 35 ml.

ethylenediamine-tetraacetic acid 30di~3~dium salt : 0~1 g.

. dioctyl sodium sulphosuccinate ~.5 g.

*trademark - 15 - ~; :

. ~ - ~ . . .

``~ llU1318 2~5-dimethylhexane-2,5-dihydro~
peroxide ~about 70~) la 6 g~
pho~phoric acid trimorpholide 12.7 g. ;-;~ :
~: ethanol 3V~0 ml. -~
distilled water ad 100 ml~
Solution 2 . . ~, ~
3,3t,5,5 t -tetramethylb~nzidine 0O3 g~
phenanthridine 0.2 g.
: l-phenyl~emicar~azide: ~ O~Q5 g~
methanol/toluene (40:60 v/v) ad 100 ml.
Test papers of this ~ind ~xq practically not influenced by light and air. Test paper~ without the 1-aryl~emicarbazide u~ed ac ording to the ~resent invention .
become green coloured after illumination ~or 5 to 10 minute~. Papers in which, in~tead of the L-aryl~emi- ;
~arbazide used according to the pre3ent invention~ an ~:
`~ equi~olar amount of aminoguanidine:or of Gemicarbazide ~ ~ haG been;introduced-~:into the impre~ation sQlution have .~ equally ~nfavouràble properties.
m c l-phenylqemicarbazide can be replaced by equi-.Lar amount~ of l~ chlorophenylj-~emucarbazide, 1~
tolyl)-~emicarbazide ~r l-(~-methoxy)-semicarbaæide, ?
wn~hout altering the propertles~o~ the paper~. Blood can . . -b~ deteeted with ~ufficient sensitivity in urine or in liquor with the paper~ produccd according to thi~ Example.
~- Example 7.

:~ ~ Filter paper (Schleicher ~ Schull 2312) iG ~ucce~s~
ively impregnated with the following 301ution~ and then :
dried: . ~

*trademark .
- 16 _ L33 ~ ~

Solution 1:
glucoqe ox:idase (55 ~J/mg. ) 500 mg.
peroxida~e (75 U/mg. ) 200 mg., tartrazine 100 mg~
di ~tilled water 100 ml .
Solution 2 ~
3, 3 ', 5, 5 ' ~tetramethylbenzidine 600 ~gJ ~ ~ , l-phenyl semicarbazide 50 mg O
~odium lauryl ~arcosinate ~ 100 mgO : -di stilled wa1:er 100 ml .
A paper produced in thi~ manner ~ stable toward~
the influence~3 of light, air and atmospheric moi~sture and, .
with gluco~e concentrations of 30 mg. to 2000 mg. per dl. ::
of urine, ~hows an increasingly irltensive green îndlcator reaction. I:, in the production of the paper, th~ l-ar~
~emicarbazide used according to the pre:3ent inventiorl i s ~ :
omitted, then paper~ are obtained which are~qensitivo to light, air a~d atmospheric humidity. Within a ~hort ~ ~;
period of time, they become discoloured 80 that, in the case of the detec~ion of glucose in urine, falsely po~itive reactions can occur.
Fxa~

Filter paper (Schleicher & Schull 231 ~ is impreg-nated with a ~olution of the following compo-~ition and then dried ~
disodium salt of 2~2'-azino-di- :
L l-e~hyl-quinoline~(2)-di- ~
3ulp~0nic acid-(6)] 100 mg. -~:
l~phenylsemucarbazide 50 mg. ,~
polyvinylpyrrolidone 2~0 mg.

*trademaxk 3~l8 ~ . :
, gluco~ oxida~e (50 U/mg. 3 400 mg~
peroxida~e (75 U/mg. ) 100 mg.

0.4 molar ~odium citra~e bu~fer, p~ 5 0 100 ml"
A paper produced in thi~ manner pe~it~ th~
detection of gluco~e concentration~ in urine above about 50 mg. ~dl " by means of a coloration toward~ violet. It i~ not di~coloured i~ lig~t and air. F~oweve~, papers which have been produce~l without the addition of the 1-aryls~micarba2ide u~ed accc: rding to the pre~ent invention b~came discoloured due to the actidn ~f light and a~r~

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Claims

The embodiments of the invention in which an exclusive property or privilege is claimed are defined as follows:

1. A diagnostic agent for the detection of hydro-peroxides or of substances which react with the liberation of hydroperoxides or of peroxidase or of peroxidate-active substances, using a stabilized oxidation indicator and using the reaction of a hydroperoxide or of a peroxidate-active substance with an oxidation indicator and evaluating the coloration produced by the reaction, wherein the stabilizer used is a 1-arylsemicarbazide of formula (I):
Ar-NH-NH-CO-NH2 (I) in which Ar is an aryl radical unsubstituted or substituted by alkyl, alkoxy or halogen.

2. A diagnostic agent according to claim 1, wherein Ar is phenyl or naphthyl, unsubstituted or substituted by alkyl of 1 to 4 carbon atoms, alkoxy of 1 to 4 carbon atoms, fluorine, chlorine or bromine.

3. A diagnostic agent according to claim 1 or 2, for the determination of glucose in body fluids, wherein the oxidation indicator is a benzidine or azine compound.

4. A diagnostic agent according to claim 1 or 2, for the determination of occult blood in faeces, wherein the oxidation indicator is guaiaconic acid A with the specific extinction E?% cm. at 600 nm of at least 200, determined by the reaction of peroxidase and hydrogen peroxide, or is a benzidine or azine compound.

5. A diagnostic agent according to claim 1 or 2, wherein the components are impregnated on to an absorbent carrier,

6. A diagnostic agent according to claim 1 or 2, wherein there is also present at least one additional adjuvant selected from buffers, wetting agents, thickening agents, protective colloids and complex formers.

7. A diagnostic test strip for rapid testing of body materials comprising an absorbent substrate impregnated with an effective amount of an oxygen indicator effective to produce a perceptible coloration on oxidation and 1-arylsemi-carbazide of formula (I) Ar-NH-NH-CO-NH2 in which Ar is an aryl radical unsubstituted or substituted by alkyl, alkoxy or halogen, in an amount effective to stabilize the oxidation indicator from coloration due to light or air.

8. A test strip according to claim 7, wherein Ar is phenyl or naphthyl, unsubstituted or substituted by alkyl of 1 to 4 carbon atoms, alkoxy o 1 to 4 carbon atoms, fluorine, chlorine or bromine.

9. A test strip according to claim 7, wherein the oxidation indicator is guaiaconic acid A with the specific extinction E?% cm. at 600 nm of at least 200, determined by the reaction of peroxidase and hydrogen peroxide, or is a benzidine or azine compound.

10. A test strip according to claim 7, wherein said sub-strate comprises a filter paper.

11. A test strip according to claim 7, 8 or 9, wherein said substrate is further impregnated with at least one additional adjuvant selected from buffers, wetting agents, thickening agents, protective colloids and complex formers.

.

12. A test strip according to claim 7, 9 or 10, wherein Ar is phenyl or naphthyl.

13. A test strip according to claim 7, 9 or 10, wherein Ar is m-chlorophenyl, p-chlorophenyl or o-tolyl.

14. A test strip according to claim 7, 9 or 10, wherein Ar is m-tolyl, p-tolyl, o-chlorophenyl or methoxyphenyl.

15. A test strip according to claim 7, 8 or 3, wherein said substrate is further impregnated with a hydroperoxide effective in the presence of peroxidate-active substance to oxidise the oxygen indicator.

16, A test strip according to claim 7, 8 or 9, for the determination of glucose in body fluids wherein said substrate is further impregnated with glucose oxidase in an amount effective to catalyze the oxidation of glucose by atmospheric oxygen, and peroxidase or a peroxidate-active substance effective to catalyze the oxidation of the oxygen indicator.

17. A test strip according to claim 7, 8 or 9, wherein said 1-arylsemicarbazide is impregnated in said substrate from an impregnation solution containing 5 to 900 mg of 1-aryl semicarbazide.

18. A method for the detection of occult blood in faeces which comprises applying a sample of faeces to a first side of a test strip of claim 7 or 8, applying hydrogen peroxide dropwise to a second side of the test strip, opposite the first side, and evaluating any coloration in the test strip, as an indication of the presence or absence of peroxidate-active blood components in said sample,

19. A method for the detection of blood in a body fluid which comprises applying a sample of said fluid to a test strip as defined in claim 7 or 8 which has been impregnated with a hydroperoxide effective in the presence of peroxidate-active substance to oxidize the oxygen indicator, and evaluating any coloration in the test strip, as an indication of the presence or absence of peroxidate-active blood components in said sample.

20. A method for the detection of glucose in a body fluid which comprises, applying a sample of said body fluid to a test strip as defined in claim 7 or 8, which has been impregnated with glucose oxidase in an amount effective to catalyze the oxidation of glucose by atmospheric oxygen, and peroxidase or a peroxidate-active substance effective to catalyze the oxidation of the oxygen indicator, and evaluating any coloration in the test strip as an indication of the presence or absence of glucose in said sample.

21, A diagnostic agent for the detection of (a) hydro-peroxides or of substances which react with the liberation of hydro-peroxides or of (b) peroxidase or of peroxidatively-active substances, comprising a stabilized oxidation indicator and, in case (a), peroxidase or a peroxidatively active sub-stance or, in case (b), hydroperoxide or a substance which reacts with the liberation of hydroperoxides wherein the stabilizer is a 1-arylsemicarbazide of the formula Ar-NH-CO-NH2 in which Ar is aryl or aryl substituted with alkyl, alkoxy or halogen.